Duchenne muscular dystrophy (DMD) is an X-linked genetic muscle disease affecting 1 of every 3,500 newborn males (Kunkel et al. Nature (London) 322, 73-77 [1986]). The progressive muscle degeneration and weakness usually confine the patients to wheelchairs by their early teens, and lead to death by their early twenties. DMD is caused by recessive mutations in the dystrophin gene, the largest gene known to date, which spans nearly 3 million base-pairs on the X-chromosome with 79 exons, a coding sequence of about 11 kb, and a high rate of de novo mutations. (Koenig et al. Cell 50, 509-517 [1987]).
Dystrophin is an enormous rod-like protein of 3,685 amino acids (aa) localized beneath the inner surface of muscle cell membrane (Watkins, S. C. et al. Nature 333, 863-866 [1988]). It functions through four major structural domains: a N-terminal domain (1-756 aa), a central rod domain (757-3122 aa), a cysteine rich (CR) domain (3123-3409aa), and a distal C-terminal domain (3410-3685 aa). The N-terminal domain binds to the F-actin of cytoskeletal structures, while the CR domain along with the distal C-terminal domain anchors to the cell membrane via dystrophin-associated protein (DAP) complexes, thus, dystrophin crosslinks and stabilizes the muscle cell membrane and cytoskeleton. The central rod domain contains 24 triple-helix rod repeats (R1-R24) and 4 hinges (H1-H4). Each repeat is approximately 109 aa long. (Koenig et al. J Biol Chem 265, 4560-4566 [1990]). The central rod domain presumably functions as a “shock absorber” during muscle contraction. Dystrophin crosslinks and stabilizes the muscle cell membrane and cytoskeleton. The absence of a functional dystrophin results in the loss of DAP complexes and causes instability of myofiber plasma membrane. These deficiencies in turn lead to chronic muscle damage and degenerative pathology.
The vast majority of DMD mutations disrupt the dystrophin mRNA reading frame or introduce a stop codon that prematurely ends protein translation (Monaco et al. Genomics 2, 90-95 [1988]). In the less severe allelic form of the disease, Becker muscular dystrophy (BMD), dystrophin gene mutations are usually such that the mRNA reading frame is maintained. Thus in BMD patients, some functional gene product, albeit of reduced quantity and/or quality, is synthesized that contributes to the milder phenotype (Hoffman et al. N. Engl. J Med. 318, 1363-1368 [1988]).
The mdx mouse (Bulfield et al. Proc. Natl. Acad. Sci. USA 81, 1189-1192 [1984]) is an animal model of DMD. The genetic lesion in the mdx dystrophin gene is a nonsense mutation at base 3185 of the mRNA that causes premature termination of translation within exon 23. This nonsense mutation precludes synthesis of a functional protein.
Due to the lack of effective treatment for DMD, novel genetic approaches including cell therapy and gene therapy have been actively explored. However, clinical trials of myoblast transplantation have met with little success owing to the poor survival of the transplanted cells (Gussoni et al., Nature Med 3, 970-977 [1997]). It was recently reported that gentamicin treatment in mdx mice led to the suppression of the premature stop codon in the dystrophin gene, and the subsequent expression and localization of functional dystrophin to the cell membrane (Barton-Davis et al. J Clin Invest. 104, 375-381 [1999]). This treatment could prove effective in up to 15% of patient with DMD.
Somatic gene transfer offers a new approach to replace the defective dystrophin gene. A preferred approach for introducing genetic material encoding a gene product into an organ or a tissue is by use of a viral vector. In this situation, the genetic material encoding the gene product is inserted into the viral genome (or a partial viral genome). The regulatory elements directing the expression of the gene product can be included with the genetic material inserted into the viral genome (i.e., linked to the gene inserted into the viral genome) or can be provided by the viral genome itself, for example, a retrovirus long terminal repeat (LTR) or an adeno-associated virus (AAV) inverted terminal repeat (ITR). Infection of cells with a viral vector has the advantage that molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid and viral vector systems can be used in vivo. Different viral vectors are described separately in the subsections below.
1. Adenovirus vectors: The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle (Curiel, Ann N Y Acad Sci 886, 158-71 [1999]). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium, endothelial cells and muscle cells. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Haj-Ahmand et al. J. Virol. 57, 267-273 [1986]). Most replication-defective adenoviral vectors currently in use are deleted for all or parts of the viral E1 and E3 genes but retain as much as 80% of the adenoviral genetic material. Adenoviral vectors deleted for all viral coding regions are also described by Kochanek et al. and Chamberlain et al. (U.S. Pat. Nos. 5,985,846 and 6,083,750).
Adenovirus vectors have been successfully tested in dystrophic animal models (Ragot et al. Nature 361, 647-50 [1993]; Howell et al. Hum Gene Ther 9, 629-34 [1998]). Nonetheless, the immunogenicity and inefficiency of infecting mature muscle cells remain major hurdles to overcome before the adenovirus vectors can be safely used in humans.
2. Herpes simplex virus (HSV) vectors: The main feature of an HSV vector is that it has very large packaging capacity, is usually replication defective and does not integrate into the host genome. HSV infects cells of the nervous system (Fink et al. Annu Rev Neurosci 19, 265-287, [1996]). The virus contains more than 80 genes, one of which (IE3) can be replaced to create the vector. The generation of HSV vectors with deletions in many of the immediate early gene products has resulted in vectors with reduced toxicity and antigenicity, as well as prolonged expression in vivo. However, these modifications also result in a lower virus yield. Construction of HSV vectors is described in U.S. Pat. No. 5,661,033.
3. Retrovirus vectors: Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (Miller A D Blood 76, 271-278 [1990]). The members of the family Retroviridae are characterized by the presence of reverse transcriptase in their virions. There are several genera included within this family, including Cisternavirus A, Oncovirus A, Oncovirus B, Oncovirus C, Oncovirus D, Lentivirus, and Spumavirus.
A recombinant retrovirus can be constructed having a nucleic acid encoding a gene product of interest inserted into the retroviral genome. Additionally, portions of the retroviral genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in “Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14” and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus cell lines include .psi.Crip, .psi.Cre, .psi.2 and .psi.Am.
Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, hematopoietic stem cells, in vitro, and/or in vivo (U.S. Pat. Nos. 4,868,116; 5,449,614 and 6,207,455). Retroviral vectors require target cell division in order to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication of the target cell. Successful transductions of hematopoietic stem or progenitor cells with retroviral vectors in an ex vivo setting have been reported. However, Recombinant retroviral vectors can only accommodate about 8 kb to 10 kb of foreign DNA. This packaging capacity also limits its use in the genetic treatment of DMD.
4. Lentivirus vectors. Lentiviruses also belong to the retrovirus family, but they can infect both dividing and non-dividing cells. The best-known lentivirus is the human immunodeficiency virus (HIV), which has been disabled and developed as a vector for in vivo gene delivery. Like the simple retroviruses, HIV has three genes termed gag, pol and env, it also carries genes for six accessory proteins termed tat, rev, vpr, vpu, nef and vif. Using the retrovirus vectors as a model, lentivirus vectors have been made, with the transgene enclosed between the LTRs and a packaging sequence (Naldni et al. Science 272, 263-267 [1996]). Some of the accessory proteins can be eliminated without affecting production of the vector or efficiency of infection.
When lentiviral vectors are injected into rodent brain, liver, muscle, eye or pancreatic islet cells, they give sustained expression for over six months. Little is known about the possible immune problems associated with lentiviral vectors. Furthermore, there seems to be no potent antibody response. A major concern about lentiviral vector is its safety in human applications. However, recent development in producing the third generation lentiviral vectors with more deletion in viral genes and improved safety may allow for the general application of lentiviral vectors to in vivo gene therapy.
5. Adeno-associated viruses (AAV) vectors: AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al. Curr. Topics in Micro. and Immunol. 158, 97-129 [1992]). AAV vector is the only viral vector system that is based on a non-pathogenic and replication defective virus. It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al. Am. J Respir. Cell. Mol. Biol. 7, 349-356 [1992]; Samulski et al. J. Virol. 63, 3822-3828 [1989]). Vectors containing as little as 300 base pairs of AAV DNA can be packaged.
AAV vectors have been successfully used to establish efficient and long-term gene expression in vivo in a variety of tissues without significant immune response or toxicity (Xiao et al. J. Virol. 70, 8098-108 [1996]; Kessler et al. Proc Natl Acad Sci USA 93, 14082-7 [1996]; Xiao et al. J Virol 72, 10222-6 [1998]). Unlike other viral vectors, AAV readily bypasses extracellular barriers due to its small viral particle size (20 nm) that facilitates efficient transduction of muscle myofibers of various maturity (Pruchnic et al. Hum Gene Ther 11, 521-36 [2000]). AAV can also be delivered into a large number of muscle groups via the blood vessels (Greelish et al. Nat. Med. 5, 439-443 [1999]) The unparalleled efficiency and safety have led to an increasing interest in AAV-mediated gene therapy for genetic muscle disorders, as well as for metabolic diseases. However, a major obstacle for AAV vectors is the limited packaging size that only allows for genes smaller than 4.7 kb (Song et al. Proc Natl Acad Sci U S A 95, 14384-8 [1998]; Kay et al. Nat Genet 24, 257-261 [2000]), therefore precludes such large gene as dystrophin with a cDNA of 14 kb.
Other viral vector systems that may have application in the subject invention have been derived from vaccinia virus (Chen et al. J Immunother 24, 46-57 [2001]), and several RNA viruses. The plus-strand RNA viridae, such as polio (Bledsoe et al. Nat Biotechnol. 18, 964-9 [2000]), hepatitis A (Romano G. Stem Cells; 18, 19-39 [2000]), and sindbis virus (Wahlfors et al. Gene Ther 7, 472-80 [2000]) are being developed for high-level gene expression, following either viral infection or delivery of nucleic acids using a nonviral system. These viruses express a replicase protein that can specifically replicate the viral RNA. By inserting a transgene in place of the viral capsid gene(s), it is possible to generate a chimeric RNA that replicates autonomously in the cell and expresses a high level of protein from the plus-coding strand of RNA. These viral vectors are well suited for immunization strategies in which high, transient gene expression is needed to induce an immune response to the transduced cells.
In addition to the viral gene transfer vectors, powerful non-viral gene transfer vectors have also become available for clinical application in the past several years (Ropert et al. Braz J Med Biol Res. 32, 163-9 [1999]; Lee R J et al. Crit Rev Ther Drug Carrier Syst 14, 173-206 [1997]). These vectors rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules to deliver genetic materials into cells. These vectors include cationic and other liposomes, DNA-viral conjugates, RNA/DNA oligonucleotides and, surprisingly, naked DNA molecules. Physical procedures, such as hydrodynamics-based and electroporation-based procedures have been used to improve gene transfer efficiency of some non-viral vectors (Zhang G. et al. Gene Ther 7, 1344-9 [2000]; Yamashita et al. Cancer Res. 61, 1005-12 [2001]). Recently, it was also reported that intraperitoneal injection of a β-galactosidase fused to the protein transduction domain from the human immunodeficiency virus TAT protein resulted in delivery of the fusion protein to all tissues in mice (Schwarze et al. Science, 3, 1569-1572 [1999]).
Somatic gene transfer using non-viral vectors carrying dystrophin gene have been attempted [Acsadi et al. Nature 352, 815-818 [1991]; Rando et al. Proc. Natl. Acad. Sci USA 97, 5363-5368 [2000]). Transgene expression was achieved with only very limited efficiency.
Previous attempts to generate dystrophin minigenes that were shorter than ½ of the full-length dystrophin failed to preserve the essential protective functions. Cox et al. and Greenberg et al. reported that expression of Dp 71, a 71 kD non-muscle product of the dystrophin gene that consists of the cysteine-rich and C-terminal domains of dystrophin (exon 63-79), in the skeletal muscle of dystrophin deficient mdx mice restored normal levels dystrophin associated proteins (DAPs). However, expression of Dp71 failed to alleviate symptoms of muscle degeneration [Cox et al. Nature Genet 8, 333-339 [1994]; Greenburg et al. Nature Genet 8, 340-344 [1994]). Similarly, Yuasa et al (Yuasa et al. FEBS Lett 425, 329-336 [1998]; Yamamoto et al. Hum Gene Ther 11, 669-80 [2000]) demonstrated that expression of dystrophin minigenes with both intact N- and C-terminal domains and 1 to 3 central rod repeats in mouse skeletal muscle was sufficient to restore DAP complexes but insufficient to restore myofiber morphology and to prevent dystrophic pathology.